Fermentation process

ABSTRACT

Increased yields of the known and useful antibiotic cephamycin C (7-(D-5-amino-5-carboxyvaleramido)-3-(carbamoyloxymethyl)-7methoxy-3-cephem -4-carboxylic acid) are obtained by the addition of D-lysine or DL-lysine to fermentation media composed of complex organic or chemically defined nutrients.

United States Patent [1 1 Inamine et al.

[ 51 Sept. 2, 1975 FERMENTATION PROCESS [75] Inventors: Edward Inamine, Rahway; Jerome Birnbaum, Morganville, both of NJ.

[73] Assignee: Merck & Co., Inc., Rahway, NJ

[22] Filed: Dec. 11, 1974 [21] Appl. N0.: 531,650

Related US. Application Data [62] Division of Ser. No. 464,007, April 25, 1974.

[52] US. Cl 195/100; 195/102 [51] Int. Cl. C12K l/10 [58] Field of Search 195/80 R, 99, 100, 101,

[56] References Cited UNITED STATES PATENTS 3,801,464 4/1974 Gorman ct a1. 195/80 R Primary Examiner-Lionel M. Shapiro Assistant ExaminerRobert J. Warden Attorney, Agent, or Firm-Julian S. Levitt; .1. Jerome Behan 5 7 ABSTRACT 3 Claims, No Drawings FERMENTATION PROCESS This is a division of application Ser. No. 464,007 filed, April 25, 1974.

This invention relates to an improved fermentation process for the production of the known and useful antibiotic cephamycin C l 7-( D-5-amino-5- carboxyvaleramido )-3-carbamoyloxymethyl )-7- methoxy-3-cephem-4-carboxylic acid]. [Antimicrobial Agent and Chemotherapy, Vol. 2, September 1972, pages 121-131, 132-135, 281-286 and 287-290.] In particular, this invention relates to an improved method for the production of the antibiotic by fermentation of nutrient media with suitable strains of microorganisms such as, for example, Streptomyces.

The antibiotic is produced during the aerobic fermentation of suitable aqueous nutrient media under controlled conditions. Aqueous media such as those employed for the production of other antibiotics are suitable. Such media contain sources of carbon and nitrogen which are assimilable by the microorganism, and inorganic salts. In addition, the fermentation media contain traces of metals necessary for the growth of the microorganism which are commonly supplied as impurities incidental to the other constituents of the medium. In general, carbohydrates such as sugars, for example, sucrose, maltose, fructose lactose, and the like, and starches such as grains, for example, oats and rye, corn starch, corn meal and the like can be used either alone or in combination as sources of assimilable carbon. The exact amount of the carbohydrate source or sources utilized in the medium will depend in part upon the other ingredients. It has been found, however, that an amount of carbohydrate between about 1 and 6 percent by weight of the medium is sufficient. A single carbon source may be used, or several carbon sources may be combined.

Satisfactory nitrogen sources include myriad proteinaceous materials such as various forms of hydrolysates of casein, soybean meal, corn steep liquor, distillers solubles, yeast products, tomato paste, and the like. The various sources of nitrogen can be used either alone or in combination, and are used in amounts ranging from 0.2-6 percent by weight of the aqueous medium.

The fermentation is carried out at temperatures ranging from C. to 37C., however, fof optimum results, it is preferable to conduct the fermentation at temperatures of from about 24C. to 32C. The pH of the nutrient mediums suitable for growing the Streptomyces cultures and producing the antibiotic should be in the range of from about 6.0 to 8.0.

Cephamycin C is produced during the aerobic fermentation described above by a strain of Streptomyces luctmmluratts and Streptomyces eluvuligerus capable of producing said Compound as, for example, by the strains on deposit in the culture collection of the Northern Utilization Research and Development Branch of the US. Department of Agriculture at Peoria, Ill. under accession numbers NRRL 3802 and NRRL 3585, respectively. Other strains of this species, such as mutants obtained by mutating agents or isolated from nature, can also be used.

Cephamycin C and its salts demonstrate resistance not only to penicillinase but to the cephalosporinases as well. This compound is active in inhibiting the growth of gram-positive and gram-negative microorganisms. Unlike cephalosporin C which has relatively low antibacterial activity, cephamycin C exhibits a significant in vivo gram-negative effect with a potency which, in general, is greater than cephalothin. This activity includes effectiveness against the following gramnegative bacteria: Escherichia coli, Proteus vulgaris, Proteus mirabilis, Salmonella schottmuelleri, Klebsiella pneumoniae AD, Klebsiella pneumoniae B, and Paracelonbacterium ttrizonae.

Bioassays for the antibiotic are run by a disc-plate procedure using inch filter paper discs, in accordance with the procedure described in US. Pat. Nos. 3,769,169 and 3,770,590, the contents of which are incorporated herein by reference. The assay plates are prepared using Difco nutrient agar plus 2.0 g./l. Difco yeast extract at 10 ml. per plate. An overnight growth of the assay organism, Vibrio percolans ATCC 8461 is diluted in sterile saline solution to a suspension having 40 percent transmittance at a wave length of 660 a. This suspension is added at 20 ml. /liter of medium prior to pouring the plates.

The assay plates are held at 4C. until used (5 day maximum). Following the application of the antibioticsaturated assay discs the plates are incubated at 28C. for a period of from 8 to 24 hours. Zones of inhibition are read as mm. diameter. They are used to determine relative potencies or, when compared with a purified reference standard, the potency in ,ug./ml.

Due to the inherent difficulty in separating pure cephamycin C from the large quantities of impurities in the fermentation broth, it is of considerable importance to find a way to increase the concentration of the antibiotic relative to the total broth solids.

It is, therefore, an object of this invention to provide a method of increasing the yield of antibiotic in a fermentation process. It is a further object of the invention to provide a method ofincreasing the yield of an antibiotic using relatively inexpensive, readily available chemical additives in the fermentation process. Further objects of the invention will become apparent.

It has been discovered that the addition of D-lysine or DL-lysine to complex organic and chemically defined fermentation media will enchance the production of cephamycin C.

By complex organic media is meant media wherein some of the ingredients are not chemically defined. An example of such media is one consisting of Crescent brand oats, soybean meal, sodium citrate, a defoamer and distillers solubles. By "chemically defined or synthetic media is meant media in which all of the ingredients are chemically defined. An example of such media is one consisting of glucose, potassium acid phosphate, sodium citrate, asparagine, methionine, monosodium glutamate, calcium chloride, magnesium sulfate and ferric sulfate.

The amount of the D-lysinc or DL-lysine needed to stimulate production of the antibiotic is to some extent dependent upon both the culture and the medium employed. In the case of the S!repmmyces Iucttumlurunx or SIreplomyc'ar t'luruligel'm' culture an increase in the production of theantibiotic has been observed in synthetic media containing from 0.0l to 0.80 percent (weight/volume) of either additive. Optimum yields are obtained at a level of 0. 10 to 0.80 percent with particularly good yields at 0.40 percent.

Correspondingly, in a complex organic nutrient medium using Streplunzycm luc'tmmlurum' or .S'trt'pmmycet clavuligerus, an increase in the yields of antibiotic are observed at a level of 0.05 to 0.20 percent of D-lysine or DL-lysine, with optimum yields obtained at 0.10 to 0.20 percent.

One skilled in the art will readily appreciate that in addition to being used singly, the D-lysine and DL- lysine may be combined to afford an additive that will stimulate the yield of cephamycin C in both complex organic nutrient media and synthetic media employing either S. luctamdurwzs or S. cluvuligerus.

One skilled in the art will further appreciate that in addition to employing D-lysine and DL-Iysine. salts of these materials may be utilized in the practice of the invention. For example, the HCl, SO -PO etc. salts may be employed in the basal production medium to increase the yield of antibiotic.

The time of addition of the yield-increasing additives to the fermentation batch is not critical. Thus, the addition may take place at the time of inoculation with the Streptomyces culture to as long as 72 hours subsequent. In general, it is preferred to add the Dlysine or DL-lysine at the time of inoculation.

The media to which the D-lysine or DL-lysine compound are added can be any suitable aqueous nutrient media; however, certain media when used in combination with these additives yield particularly good amounts of the antibiotic. Thus, the Streptomyces c'luvuligerus culture has been found to afford maximum yields in the following medium:

MEDIUM l Starch-4.8%

Distillers Solubles().5%

Soya Bean Grits0.21%

Glycerol0.8%

Hydrolyzed Casein (*N-Z Amine, Type A)O.5%

Ferrous Sulfate Heptahydrate0.0 1 7r Tap Water-10 liter *N-Z Amine. Type A: an enzyme digest ofcasein; product of the Shel field Chemical Co. Norwich. N.Y.

The Srrcptonrvccs lactumduruns culture affords especially good yields of antibiotic in Medium ll infra:

MEDIUM ll Distillers So]ubles3.0%

Primary Dried Yeast 1.0%

Mobile par-S Defoamer0.25%

Glycine0.05%

L-phenylalanine 0.30%

Cornstarch2.0%

Deionized Waterl .0 liter The above discussion and examples below are primarily directed to fermentations using a particular strain of the Strepumyvces lac'tunulumns and Streptonuces clavuligerus cultures. However, other strains of these organisms such as mutants can also be used to produce the antibiotic and it should be obvious to one skilled in the art that DL-lysine or Dlysine can be used to in crease the antibiotic yield. when added to fermentation batches containing such strains. Following the teaching of this invention obvious modifications or changes in the optimal levels of the additive or the time of addition to the fermentation medium, etc., will be within the skill of the artisan, no matter which strain of Streptomyces [aclamdumns or .S'lrepmnrvces clm'ull'gcrux is used to produce the antibiotic cephamycin C.

Although the antibiotic is produced by both surface and submerged cultures, it is presently preferred to carry out the fermentation in the submerged state. Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in flasks, sterilizing the flasks and contents by heating to C. inoculating the flasks with either spores or a vegetative cellular growth of a cephamycin C produc ing strain of Streptomyces, loosely stoppering the necks of the flasks with cotton, and permitting the fermentation to proceed at a constant temperature of about 28C. on a shaker for 3-5 days. For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means for aerating the fermentation medium. In this method, the nutrient medium is made up in the tank and sterilized by heating at 120C. After cooling the sterilized medium is inoculated with a suitable source of vegetative cellular growth of the Streptomyces culture and the fermentation is permitted to proceed for 2-4 days while agitating and/or aerating the nutrient medium and maintaining the cephamycin C is particularly suited for the preparation of large quantities of the antibiotic.

The fermentation using the cephamycin C producing microorganism can be carried out at temperatures ranging from about 2037C. For optimum results, however, it is most convenient to conduct the fermentation at temperatures between 2630C. The pH of the nutrient media suitable for growing the Streptomyces and producing the antibiotic may vary from about 5 to 9. The preferred pH range, however, is from about 6.0 to 7.5.

In carrying out the invention, a cell suspension is prepared by the addition of sterile medium to an agar slant culture of the cephamycin C producing microorganism. Growth from the slant culture is then used to inoculate a seed flask and the seed flask is shaken at about 28C. for l3 days in order to obtain good growth. The seed flask is then used to inoculate the production flasks. Alternatively, the seed flask can be inoculated from a lyophilized culture or a frozen inoculum.

The inoculation is generally carried out using about 1 ml. per 40 ml. of production medium. The desired concentration of additive is then added to the production flasks after the necessary time period of waiting and the fermentation is permitted to proceed for 2-4 days while agitating and/or aerating the nutrient medium and maintaining the temperature at about 28C. All of the production flasks, i.e., those containing additives and the flasks used as controls, are then assayed, generally after 96 hours, to determine the amount of antibiotic produced in each flask.

The antibiotic is conveniently assayed by means of a disc-plate procedure using Vibrio percolans ATCC 846l as the assay organism. Discs of /8 inch diameter are used. Activity of antibiotic is expressed in terms of .Lg. per ml. of the free acid. A standard curve is prepared from known concentration solutions of the antibiotic.

The product on flasks are then assayed by diluting the sample in 0.02 molar phosphate buffer at pH 7 to an appropriate concentration. The test organism is Vibrio percolum' ATCC 8461, and the assay medium is Difco nutrient agar plus 0.2 percent Difco yeast extract. The discs are dipped into 5 pg per milliliter of the standard antibiotic solution and are placed on the plate in a position alternate to the sample. The plates are then incubated at 37C. for 18 hours, and the zone diameters in millimeters are determined. Five standard plates containing 4 levels of the standard ranging from 2.5 pig/ml. to 20 ,agjml. are employed. The assay is calculated by means of a Nomograph. and the results are reported in terms of ag. per milliliter of the free acid.

The antibiotic can be recovered from the fermentation medium by a number of procedures. The filtered broth can be passed through one or more ion exchange columns. The amphoteric nature of the antibiotic enables selection of both cationic and anionic ion exchange resins to optimize recovery. The absorbed antibiotic can then be removed by elution, preferably in a volatile solvent such as pyridine which can be easily removed.

The following examples are given for purposes of illustration and not by way of limitation.

EXAMPLE I A lyophilized tube of S. lac'lamduranx NRRL 3802 was opened aseptically and its contents transferred to a slant culture tube with an agar maintenance medium of the following composition:

Blackstrap molasses Brewers Yeast (National Yeast Co.) Bacto-Agar (Dil'co) pH 7.0

After inoculation the slants were incubated at 28C. for 7 days at which time abundant growth was apparent. The cultures were stored at refrigerator temperatures until use.

First Stage Seed: A loopful of the organism from the slant culture was transferred to 40 ml. of sterile first stage seed medium contained in a 250 ml. baffled Erlenmeyer flask. The medium containing 10 g/l Primary Dried Yeast, N.F. (supplied by Yeast Products Co., Paterson. NJ.) in deionized water. The flask was incubated at 28C. on a rotary shaker (2-inch displacement) set at 220 rpm, for 48 hours, at which time luxuriant growth of the organism was apparent.

Second Stage Seed: One ml. ofthe first seed was used to inoculate 40 ml. of 2% Fleishmann S-l5O Yeast Autolysate pH 7.0 contained in a 250 ml. baffled Erlenmeyer flask. This nutrient is a product of the Fleischmann Yeast Co., Stamford. Conn. The second stage seed was incubated as described above for 48 hours.

Basal Production Medium: The basal production medium has the following composition:

8/ Distillers Solubles (Brown & Forman Co.) 30 Primary Dried Yeast (Yeast Products Co.) 7.5 Mohil Par-S Dcfoamer 2.5 Sodium Thiosulfate" 1.0 Deionized water to I000 ml Added from a sterililetl concentrated stock solution at 30 hours post-inoculation.

The pH of the medium was adjusted to 7.0 with small amounts of sodium hydroxide solution, dispensed (40 ml.) into 250 ml. Erlenmeyer flasks and sterilized at 120C. for minutes. After cooling the medium was inoculated with 2.5% by volume of the fermented second stage seed. The incubation was for 72 hours at 28C. on a rotary shaker as described above. At 30 hours post-inoculation. a small quantity of sterile concentrated Na S O solution was added to a final level of 1.0 g/l of Na S O To a series of flasks, prepared as described above. was added L-, D-, or DL-lysine.HCl. The flasks were identical in every way except for the presence of the amino acid. The lysine solution was prepared by dissolving L, D- or DL-lysine.HCl in water and neutraliz ing to pH 7.0 with sodium hydroxide solution. These solutions were sterilized separately from the basal production medium, and aliquots were added first prior to inoculation with the production organism to give a final concentration of 0.1% in the case of L- and D-lysine and 0.2% in the case of DL-lysine.

Upon completion of the fermentation the cells were removed by centrifugation and the clarified broth assayed for cephamycin C in the manner discussed above.

First Seed Stage: A lyophilized tube of S. lactamduruns NRRL 3802 was opened aseptically and its contents transferred to 40 ml. of sterile first stage seed medium contained in a 250 ml. baffled Erlenmeyer flask. The medium contained 10 g/l Primary Dried Yeast, N.F. (supplied by Yeast Products Co., Paterson. NJ.) in deionized water. The flask was incubated at 28C. on a rotary shaker (2-inch displacement) set at 220 rpm, for 48 hours. at which time luxuriant growth of the or ganism was apparent.

Second Stage Seed: One ml. of the first seed was used to inoculate 40 ml. of medium containing 1% Ardamine YEP Yeast Autolysate (Yeast Products Co., NJ.)

at pH 7.0, in a 250 ml. baffled Erlenmeyer flask. Incubation was on a rotary shaker (2-inch displacement, 220 rpm) at 28C. for 22 hours.

Basal Production Medium: The basal production medium has the following composition:

Distillers Soluhles 3.0 w/v Primary Dried Yeast N.F. 0.75 wj'v Cornstarch (Clinton) 2.0 w/v Dimethylformamide 1.0 v/v Glycine 0.05 w/v l.-phenylalanine 0.3 w/v Mobil Par S-Defoamer 0.25 w/v Sodium 'lhiosulfate 0.1 w/v *Added at 30 hours mist-inoculation from a sterile concentrated stock solution to give the t'mal concentration as shown.

The medium was made up in deionized water, adjusted to pH 77.5 with sodium hydroxide, dispensed (40 ml.) into 250 ml. Erlenmeyer flasks and sterilized by autoclaving for 20 minutes. After cooling the medium was inoculated with 2.5% by volume of the second stage seed and incubated for 96 hours at 28C. on a rotary shaker (2-inch displacement) set at 220 rpm. The sodium thiosulfate was added at 30 hours postinoculation.

To a series of flasks, prepared as described above, was added L-, D-, or DL-lysine.HCl. The flasks were identical in every way except for the presence of the amino acid. The lysine solution was prepared by dissolving L-, D- or DL-lysine.HCl in water and neutralizing to pH 7.0 with sodium hydroxide solution. These solutions were sterilized separately from the basal production medium, and aliquots were added first prior to inoculation with the production organism to give a final concentration of 0.05 to 0.2 percent of L-, D-, or DL- lysine hydrochloride.

Upon completion of the fermentation the cells were removed by eentrifugation and the clarified broth assayed for cephamycin C in the manner described above.

EXAMPLE 111 First Seed Stage: A lyophilized tube of S. lacramduruns NRR] 3802 was opened aseptically and its contents transferred to 40 ml. of sterile first stage seed medium contained in a 250 ml. baffled Erlenmeyer flask. The medium contained g/l Primary Dried Yeast, N.F. (supplied by Yeast Products Co., Paterson, NJ.) in deionized water. The flask was incubated at 28C. on a rotary shaker (2-inch displacement) set at 220 rpm. for 48 hours, at which time luxuriant growth of the organism was apparent.

Second Stage Seed: One ml. of the first seed was used to inoculate 40 ml. of medium containing 1% Ardamine YEP Yeast Autolysate (Yeast Products Co., NJ.) at pH 7.0, in a 250 ml. baffled Erlenmeyer flask. Incubation was on a rotary shaker (2-inch displacement, 220 rpm) at 28C. for 22 hours.

Basal Production Medium: The basal production medium has the following composition:

Distillers Solubles 3.0 w/v Primary Dried Yeast N.F. 0.75 w/v Glycerol 1.0 w/v Dimethylformamide 1.0 v/v Glycine 0.05 w/v l-phcnylalaninc 0.3 \v/v Mobil Par S-Del'oamer 0.25 w/v Sodium 'l hiosull atc 0.1 w/v Added at 30 hours post-inoculation from a sterile concentrated stock solution to give the final concentration as shown.

To a series of flasks, prepared as described above, was added L-, D-, or DL-lysine.HCl. The flasks were identical in every way except for the presence of the amino acid. The lysine solution was prepared by dissolving L-, D- or DL-lysine.HCl in water and neutralizing to pH 7.0 with sodium hydroxide solution. These solutions were sterilized separately from the basal production medium, and aliquots were added first prior to inoculation with the production organism to give final concentrations of 0.05 percent to 0.2 percent of L, D-, or DL-lysine hydrochloride.

Upon completion of the fermentation the cells were removed by centrifugation and the clarified broth assayed for cephamycin C in the manner discussed above.

First Seed Stage: A lyophilized tube of S. Iaztumdurams NRRI 3802 was opened aseptically and its contents transferred to 40 ml. of sterile first stage seed medium contained in a 250 ml. baffled Erlenmeyer flask. The medium contained 10 g/l Primary Dried Yeast, N.F. (supplied by Yeast Products Co., Paterson, NJ.) in deionized water. The flask was incubated at 28C. on a rotary shaker (2-inch displacement) set at 220 rpm, for 48 hours, at which time luxuriant growth of the organism was apparent Second Stage Seed: One ml. of the first seed was used to inoculate 40 ml. of synthetic medium which was identical to the synthetic production medium described below. Incubation was in Erlenmeyer flasks at 28C. on a rotary shaker (Z-inch displacement) set at 220 rpm for 48 hours.

Synthetic Production Medium:

Glucose 1.0 Monosodium glutamate 0.425 i-lnositol 0.02 p-Aminobenxoic acid 0.000001 K HP0, 0." NH Cl 0.1 NaCl 0.05 MgSO .7H. ,O 0.05 CaCO 0.025 FeSO,.7H. .O 0.002s ZnSO,.7H. .O 0.00l MnSO H O 0.0005 Na .s ,0 0.l

Added from a sterile concentrated stock solution at 30 hours post-inoculation to give the final concentration as \'ht lL The medium was prepared with deionized water and adjusted to pH 7.0 with sodium hydroxide and 250 ml. Erlenmeyer flasks containing 40 ml. each of the medium were sterilized by autoclaving for 20 minutes.

After cooling each flask was inoculated with 1.0 ml. of the second seed. Incubation was for 96 hours at 28C. on a rotary shaker. The sodium thiosulfate was added as described above at 30 hours after inoculation. 1 D- or DL-lysine.l-ICL was added to the flasks from separately sterilized stock solutions just prior to inoculation with the organism in order to give several levels of the amino acid. Assays of antibiotic production were performed as described previously.

TABLE 4 Additions to the Cone. (Cale. Cephamycin Basal Synthetic as Hydro- C Production Production Medium chloride) ag/m1.)

None (Control) 80 D-lysine 0.01% 102 0.10% 180 0.20% 219 0.4071 270 0.80% 251 DL-lysine 0.019? 1 13 0.1071 140 0.2071 129 0.407! 148 0.8071 166 L-Iysine 0.01% 68 0.107: I 12 0.20% 65 0.4071 83 0.80% 95 EXAMPLE V Seed Stage: A lyophilized tube of Streptomyces cluvuligerux NRRL 35 85 was opened aseptically and its contents transferred into 40 ml. of a medium with the following composition:

Glucose 1.5 Soya Bean Meal 1.5 Corn Steep Liquor Solids 0.5 Calcium Carbonate ()2. Sodium Chloride 0.5 Deionized Water to 1000 mlv The pH of the solution was adjusted to 6.7 with sodium hydroxide, dispensed (40 ml.) into 250 baffled Erlenmeyer flasks and sterilized at 120C. for minutes. The inoculated flask was incubated at 28C. on a rotary shaker (2-inch displacement) set at 220 rpm for 48 hours. at which time heavy growth of the organism was apparent.

Basal Production Medium: The basal production medium has the following composition:

Cornstarch (Clinton Corn Products) 4.8 Distillers Solubles 0.5 Soya Bean Meal 0.1 Glycerol 0.8 Hydrolyzed Casein (N-Z-Amine.Type A) 0.5 Ferrous Sulfate Heptahydrate 0.0] Sodium Thiosulfate* 0.05 Tap Water to 1000 ml.

Added at start of fermentation from a sterile concentrated stock solution to give the final concentration shown.

The solution was adjusted to pH 6.5 with sodium hydroxide, dispensed (40 ml.) into 250 ml. Erlenmeyer flasks and sterilized for 20 minutes at C. After cooling the medium, sodium thiosulfate was added as described above and the flasks were inoculated with 2.5% by volume of the seed stage. The incubation was for 96 hours at 28C. on a rotary shaker as described above.

As in Example IV above, several flasks received either D- or DL-lysine.HCl. The production of antibiotic was assayed as described previously.

It is readily apparent from a consideration of Examples l-5 that D-lysine dramatically stimulate the antibiotic production of cephamycin C, whereas L-lysine does not. In fact, the L-lysine may conceivably be considered as slightly inhibitory when added to complex production media at the higher levels. The stimulation of the production of the B-lactam antibiotic cephamycin C by the unnatural amino acids D- and DL-lysine is a unique and unexpected phenomenon.

Any departure from the above description which conforms to the present invention is intended to be included within the scope of the claims.

What is claimed is:

l. A fermentation nutrient media for use in the production of cephamycin C which contains D-lysine or DL-lysine in an amount sufficient to enhance the production of cephamycin C and a cephamycin C producing specie of Streptomyccs.

2. The nutrient media of claim 1 which is a complex organic nutrient medium.

3. The nutrient media of claim 1 which is synthetic.

=l =l =l l l 

1. A FERMENTATION NUTRIENT MEDIA FOR USE IN THE PRODUCTION OF CEPHAMYCIN C WHICH CONTAINS D-LYSINE OR DL-LYSINE IN AN AMOUNT SUFFICIENT TO ENHANCE THE PRODUCTION OF CEPHAMYCIN C AND A CEPHAMYCIN C PRODUCING SPECIE OF STERPTOMYCES.
 2. The nutrient media of claim 1 which is a complex organic nutrient medium.
 3. The nutrient media of claim 1 which is synthetic. 